Genome-Wide Association Study of QTLs Conferring Resistance to Bacterial Leaf Streak in Rice.

Bacterial leaf streak (BLS) is a devastating rice disease caused by the bacterial pathogen, Xanthomonas oryzae pv. oryzicola (Xoc), which can result in severe damage to rice production worldwide. Based on a total of 510 rice accessions, trialed in two seasons and using six different multi-locus GWAS methods (mrMLM, ISIS EM-BLASSO, pLARmEB, FASTmrMLM, FASTmrEMMA and pKWmEB), 79 quantitative trait nucleotides (QTNs) reflecting 69 QTLs for BLS resistance were identified (LOD > 3). The QTNs were distributed on all chromosomes, with the most distributed on chromosome 11, followed by chromosomes 1 and 5. Each QTN had an additive effect of 0.20 (cm) and explained, on average, 2.44% of the phenotypic variance, varying from 0.00-0.92 (cm) and from 0.00-9.86%, respectively. Twenty-five QTNs were detected by at least two methods. Among them, qnBLS11.17 was detected by as many as five methods. Most of the QTNs showed a significant interaction with their environment, but no QTNs were detected in both seasons. By defining the QTL range for each QTN according to the LD half-decay distance, a total of 848 candidate genes were found for nine top QTNs. Among them, more than 10% were annotated to be related to biotic stress resistance, and five showed a significant response to Xoc infection. Our results could facilitate the in-depth study and marker-assisted improvement of rice resistance to BLS.

Transcriptome analysis of xa5-mediated resistance to bacterial leaf streak in rice (Oryza sativa L.).

Bacterial leaf steak (BLS) caused by Xanthomonas oryzae pv. oryzicola (Xoc) is a devastating disease in rice production. The resistance to BLS in rice is a quantitatively inherited trait, of which the molecular mechanism is still unclear. It has been proved that xa5, a recessive bacterial blast resistance gene, is the most possible candidate gene of the QTL qBlsr5a for BLS resistance. To study the molecular mechanism of xa5 function in BLS resistance, we created transgenic lines with RNAi of Xa5 (LOC_Os05g01710) and used RNA-seq to analyze the transcriptomes of a Xa5-RNAi line and the wild-type line at 9 h after inoculation with Xoc, with the mock inoculation as control. We found that Xa5-RNAi could (1) increase the resistance to BLS as expected from xa5; (2) alter (mainly up-regulate) the expression of hundreds of genes, most of which were related to disease resistance; and (3) greatly enhance the response of thousands of genes to Xoc infection, especially of the genes involved in cell death pathways. The results suggest that xa5 is the cause of BLS-resistance of QTL qBlsr5a and it displays BLS resistance effect probably mainly because of the enhanced response of the cell death-related genes to Xoc infection.

Genome-wide identification and expression analysis of the PHD-finger gene family in Solanum tuberosum.

Plant homeodomain (PHD) proteins are prevalent in eukaryotes and play important roles in plant growth, development and abiotic stress response. In this study, the comprehensive study of the PHD family (StPHD) was performed in potato (Solanum tuberosum L.). Seventy-two PHD genes (named StPHD1-72) were identified and grouped into 10 subfamilies based on phylogenetic analysis. Similar structure organizations were found within each subfamily according to the exon/intron structures and protein motif analysis. These genes were unequally scattered on the chromosomes of potato, with 9 pairs of segmental duplicated genes and 6 pairs of tandem duplicated genes showing that both segmental duplicated and tandem duplicated events contributed to the expansion of the potato PHD family. The gene ontology (GO) analysis suggests that StPHD mainly functioned at the intracellular level and was involved in various binding, metabolic and regulation processes. The analysis of expression patterns of StPHD genes showed that these genes were differentially expressed in 10 different tissues and responded specifically to heat, salt and drought stress based on the FPKM (Fragments per kilobase of transcript per million mapped reads) values of the RNA-seq data. Furthermore, the real-time quantitative PCR for 12 selected StPHD genes revealed the various levels of gene expression corresponding to abiotic stress. Our results provide useful information for a better understanding of PHD genes and provide the foundation for additional functional exploration of the potato PHD gene family.

[Pyramiding of 3-resistant-gene to improve rice blast resistance of a restorer line, Fuhui 673].

To improve the blast resistance of elite rice restorer line Fuhui 673, 3 blast resistance genes Pi-1, Pi-9 and Pi-kh were introduced into Fuhui 673 from a good-quality restorer line Jinhui 1059 through 3 successive backcrosses followed by one selfing using the technique of marker-assisted selection. Ten near-isogenic lines (NILs) of Fuhui 673 carrying the 3 introduced resistance genes were created. Genotype analysis using 68 SSR markers evenly distributed in the genome indicated that 92.96%-98.59% of the NILs' genetic background had been recovered to Fuhui 673. Both indoor and field resistance tests indicated that the NILs and their hybrids with sterile line Yixiang A were all resistant to rice blast, with resistance levels significantly higher than those of controls Fuhui 673 and hybrid Yiyou 673 (Yixiang A  Fuhui 673). In addition, among the 10 hybrids between the NILs and Yixiang A, 2 showed significantly higher yield than and 4 displayed similar yield to that of control Yiyou 673, suggesting that most of the NILs retained the elite characteristics of Fuhui 673. Two new hybrid rice cultivars Liangyou 7283 and Jintaiyou 683 from NIL Line 9 showed high yield, good resistance to blast and moderate growth period in regional trial, suggesting that the NIL Line 9 has a good prospect for application.

Utility of I-SceI and CCR5-ZFN nucleases in excising selectable marker genes from transgenic plants.

OBJECTIVES: Removal of selection marker genes from transgenic plants is highly desirable for their regulatory approval and public acceptance. This study evaluated the use of two nucleases, the yeast homing endonuclease, I-SceI, and the designed zinc finger nuclease, CCR5-ZFN, in excising marker genes from plants using rice and Arabidopsis as the models. RESULTS: In an in vitro culture assay, both nucleases were effective in precisely excising the DNA fragments marked by the nuclease target sites. However, rice cultures were found to be refractory to transformation with the I-SceI and CCR5-ZFN overexpressing constructs. The inducible I-SceI expression was also problematic in rice as the progeny of the transgenic lines expressing the heat-inducible I-SceI did not inherit the functional gene. On the other hand, heat-inducible I-SceI expression in Arabidopsis was effective in creating somatic excisions in transgenic plants but ineffective in generating heritable excisions. The inducible expression of CCR5-ZFN in rice, although transmitted stably to the progeny, appeared ineffective in creating detectable excisions. Therefore, toxicity of these nucleases in plant cells poses major bottleneck in their application in plant biotechnology, which could be avoided by expressing them transiently in cultures in vitro.

Toward the positional cloning of qBlsr5a, a QTL underlying resistance to bacterial leaf streak, using overlapping sub-CSSLs in rice.

Bacterial leaf steak (BLS) is one of the most destructive diseases in rice. Studies have shown that BLS resistance in rice is quantitatively inherited, controlled by multiple quantitative trait loci (QTLs). A QTL with relatively large effect, qBlsr5a, was previously mapped in a region of ∼ 380 kb on chromosome 5. To fine map qBlsr5a further, a set of overlapping sub-chromosome segment substitution lines (sub-CSSLs) were developed from a large secondary F2 population (containing more than 7000 plants), in which only the chromosomal region harboring qBlsr5a was segregated. By genotyping the sub-CSSLs with molecular markers covering the target region and phenotyping the sub-CSSLs with artificial inoculation, qBlsr5a was delimited to a 30.0-kb interval, in which only three genes were predicted. qRT-PCR analysis indicated that the three putative genes did not show significant response to the infection of BLS pathogen in both resistant and susceptible parental lines. However, two nucleotide substitutions were found in the coding sequence of gene LOC_Os05g01710, which encodes the gamma chain of transcription initiation factor IIA (TFIIAγ). The nucleotide substitutions resulted in a change of the 39th amino acid from valine (in the susceptible parent) to glutamic acid (in the resistant parent). Interestingly, the resistant parent allele of LOC_Os05g01710 is identical to xa5, a major gene resistant to bacterial leaf blight (another bacterial disease of rice). These results suggest that LOC_Os05g01710 is very possibly the candidate gene of qBlsr5a.

[Genetic analysis and gene mapping for a salt tolerant mutant at seedling stage in rice].

A salt tolerant mutant at seedling stage was obtained from an M2 population of radiation mutagenesis of an indica rice cultivar R401. The mutant seedlings could survive under the treatment of sodium chloride solution at the concentration of 150 mmol/L, while the wild-type control seedlings withered and died. An F2 population was developed from a cross between a japonica cultivar Nipponbare and the salt tolerant mutant. By investigating the performance of the F2 population under the stress of 150 mmol/L NaCl solution, we found that the mutant phenotype was caused by the recessive mutation of a single gene, temporarily designated SST(t). Bulked segregant analysis (BSA) based on the F2 mapping population revealed that SST(t) is located on chromosome 6. By analyzing 137 typical salt-tolerant F2 plants using molecular markers, SST(t) was mapped in a 2.3 cM (or 406 kb) interval between InDel markers ID26847 and ID27253, with genetic distances of 1.2 cM and 1.1 cM to the two markers, respectively.

Dwarf and deformed flower 1, encoding an F-box protein, is critical for vegetative and floral development in rice (Oryza sativa L.).

Recent studies have shown that F-box proteins constitute a large family in eukaryotes, and play pivotal roles in regulating various developmental processes in plants. However, their functions in monocots are still obscure. In this study, we characterized a recessive mutant dwarf and deformed flower 1-1 (ddf1-1) in Oryza sativa (rice). The mutant is abnormal in both vegetative and reproductive development, with significant size reduction in all organs except the spikelet. DDF1 controls organ size by regulating both cell division and cell expansion. In the ddf1-1 spikelet, the specification of floral organs in whorls 2 and 3 is altered, with most lodicules and stamens being transformed into glume-like organs and pistil-like organs, respectively, but the specification of lemma/palea and pistil in whorls 1 and 4 is not affected. DDF1 encodes an F-box protein anchored in the nucleolus, and is expressed in almost all vegetative and reproductive tissues. Consistent with the mutant floral phenotype, DDF1 positively regulates B-class genes OsMADS4 and OsMADS16, and negatively regulates pistil specification gene DL. In addition, DDF1 also negatively regulates the Arabidopsis LFY ortholog APO2, implying a functional connection between DDF1 and APO2. Collectively, these results revealed that DDF1, as a newly identified F-box gene, is a crucial genetic factor with pleiotropic functions for both vegetative growth and floral organ specification in rice. These findings provide additional insights into the molecular mechanism controlling monocot vegetative and reproductive development.

[Genetic analysis and gene mapping of DDF1, a pleiotropic gene involving in both vegetable and reproductive growth in rice].

There are many pleiotropic genes playing key roles in regulating both vegetative growth and reproductive development in plants. A dwarf mutant of rice with deformed flowers, named as ddf1, was identified from indica rice breeding lines. Genetic analysis indicated that ddf1 was resulted from the recessive mutation of a single gene, temporarily named as DDF1. This result suggested that DDF1 is a pleiotropic gene, which controls both vegetative growth and reproductive development in rice. To map this gene, an F2 population was developed by crossing the ddf1 heterozygote with the tropical japonica rice variety DZ60. By means of bulked segregant analysis and small population-based linkage analysis using the published RM-series rice SSR markers, DDF1 was preliminarily mapped in a region between markers RM588 and RM587 on chromosome 6 with the genetic distances of 3.8 cM and 2.4 cM to the two markers, respectively. By developing new SSR markers in this interval according to the published rice genome sequence, we further mapped DDF1 in a 165 kb interval. The results will facilitate cloning of DDF1.

[Towards the positional cloning of a spikelet identity gene frizzle panicle (FZP) in rice (Oryza sativa L.)].

FZP is a key gene for spikelet differentiation in rice. Mutation of the gene blocks the differentiation of spikelets and makes rachis branches develop unlimitedly. A mutant of the gene named frizzle panicle (fzp) was previously found from the high-generation progeny of a cross between two Oryza sativa ssp. indica rice varieties, V20B and Hua1B. With the mutant, FZP had been mapped to a chromosomal region of about 26.4 cM in width between two SSR (Simple Sequence Repeat) markers, RM172 and RM18, on chromosome 7. In this study, high-resolution mapping of the gene was carried out for the positional cloning of the gene. Two flanking SSR markers, NRM6 and NRM8, were identified, which are 0.2 cM and 1.0 cM apart from the target gene, respectively, bracketing the target gene within an interval of 1.2 cM or 144 kb. An APETALA2 (AP2)-domain like gene was found at the expected position of FZP. As AP2 is known to play an important role in the floral development, we took it as the most possible candidate of FZP. PCR analysis showed that the mutant allele of the AP2-domain like gene contains an insert of about 4 kb in length, suggesting that the gene is very likely FZP.